Use of interleukin-15

ABSTRACT

The invention relates to the use of IL-15 or active variants thereof and/or IL-15 activity enhancing compounds for the manufacture of a pharmaceutical composition for manipulating memory cells of the immune system, such as manipulating viability and/or responsiveness of said memory cells. The IL-15 activity enhancing compound is for example lipopolysaccharide (LPS). The invention further relates to the use of IL-15 inhibiting or eliminating compounds for the manufacture of a pharmaceutical composition for manipulating memory cells of the immune system. Such inhibiting or eliminating compounds are for example anti-IL-15 antibodies, anti-IL-15Rα antibodies, fragments of these antibodies, e.g. the Fab or F(ab′) 2  fragment, soluble IL-15Rα, fusion proteins consisting of soluble IL-15Rα, and Fc fragment, compounds, e.g. peptides, binding and/or inhibiting functional IL-15 receptor, IL-15 antisense oligonucleotides.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/045,185, filed Oct. 18, 2001, which is a divisional application ofU.S. Ser. No. 09/380,049, filed Aug. 23, 1999, now U.S. Pat. No.6,344,192, which is the U.S. national phase under 35 U.S.C. § 371 ofInternational Application No. PCT/EP98/00127, filed Feb. 23, 1998. Allof the above applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a new use of Interleukin-15 (IL-15).The invention further relates to pharmaceutical preparations, containingIL-15 itself, IL-15 stimulating compounds or IL-15 inhibiting and/oreliminating compounds.

The cytokine interleukin-15 (IL-15) was originally identified in culturesupernatants of the simian kidney epithelial cell line CV-1/EBNA and theT cell leukemia cell line HuT-102 (Grabstein et al., 1994; Burton etal., 1994; Bamford et al., 1994). The IL-15 cDNA sequence encodes a 162amino acid (aa) precursor protein consisting of a 48 aa peptide and a114 aa mature protein (Grabstein et al., 1994). Although there is nosequence homology with IL-2, analysis of the amino acid sequencepredicts that IL-15, like IL-2, is a member of the four α helix bundlecytokine family. Furthermore IL-15 and IL-2 exert their biologicalactivities through binding on the IL-2Rβ and .gamma.chains, supplementedby a specific IL-15Rα and IL-2α polypeptide, respectively (Giri et al.,1995). This sharing of receptor subunits probably accounts for thesimilar functional activities of both cytokines observed on T, B and NKcells. IL-15 mRNA is widely distributed in fibroblasts, epithelial cellsand monocytes but not in resting or activated T cells, the predominantsource of IL-2.

IL-15 and IL-2 share various biological functions. IL-15, like IL-2, hasbeen defined as a T cell growth factor. IL-15 was originally discoveredas a factor that could induce proliferation of the IL-2 dependent murinecytotoxic T-cell line (CD8⁺) CTLL-2 (Grabstein et al., 1994).Proliferation upon addition with IL-15 was also observed inphytohaemagglutinin (PHA)-activated CD4⁺ or CD8⁺ human peripheral bloodT lymphocytes (PBT), and γδ subsets of T cells (Grabstein et al., 1994;Nishimura et al., 1996). Studies with phenotypically memory (CD45RO⁺)and naive (CD45RO⁻) T cells, isolated from human PBT, revealed thatIL-15, like IL-2, induces in memory CD4⁺ and CD8⁺ T cells and naive CD8⁺T cells but not in naive CD4⁺ T cells the expression of the CD69activation marker and proliferation (Kanegane et al., 1996). IL-15 wasas effective as IL-2 in the in vitro generation of alloantigen-specificcytotoxic T cells in mixed lymphocyte cultures and in promoting theinduction of lymphokine activated killer (LAK) cells (Grabstein et al.,1994). Additionally, in vivo studies in a murine model demonstrated thecapacity of IL-15 to augment CD8⁺ T-cell-mediated immunity againstToxoplasma gondii infection (Khan and Kasper, 1996). Here vaccination ofmice with soluble parasite antigen (Ag) and IL-15 resulted insignificant proliferation of splenocytes expressing the CD8⁺ phenotypeand protection against a lethal parasite challenge for at least 1 monthpostimmunization.

Natural Killer (NK) cells are considered an important target for IL-15action. Treatment of NK cells with IL-15 results in proliferation andenhancement of cytotoxic activity and in production of Interferon γ(IFNγ), tumor necrosis factor α (TNFα) and granulocyt-macrophage colonystimulating factor (GM-CSF) (Carson et al., 1994). Furthermore IL-15 cansubstitute for the bone marrow microenvironment during the maturation ofmurine NK1.1⁺ cells from nonlytic to lytic effector cells (Puzanov etal., 1996).

Apart from its activities on T and NK cells, IL-15 costimulates, in acomparable way as IL-2, proliferation of B cells activated withimmobilized anti-IgM or phorbol ester (Armitage et al., 1995).Stimulation of B cells with a combination of CD40L and IL-15 efficientlyinduces immunoglobulin synthesis. IL-15 has no stimulatory activity onresting B cells.

IL-15 was also found to have other biological activities.Chemoattractant factors are cytokines or chemokines that regulate themigration of lymphocytes to inflammation regions.

IL-15 is described as a chemoattractant factor for human PBT, inducingpolarisation, invasion of collagen gels and redistribution of adhesionreceptors (Wilkinson and Liew, 1995; Nieto et al., 1996). Murine mastcells proliferate in response to IL-15, but not to IL-2, using a novelreceptor/signalling pathway, not shared with IL-2 (Tagaya et al., 1996).Furthermore, it has been shown that IL-15 and IL-2 have differenteffects on differentiation of bipotential T/NK progenitor cells, withIL-15 predominantly promoting the development of TCRγδ T cells and NKcells (Leclercq et al., 1996). The most striking difference, however,between IL-15 and IL-2 lies in their expression patterns. The presenceof IL-15 mRNA in a variety of non-lymfoid tissues indicates that thesecretion of the cytokine is not solely regulated by the immune systemand/or that the cytokine can act outside the immune system itself.Accordingly, addition of IL-15 to a myoblast cell line affectsparameters associated with skeletal muscle fiber hypertrophy, suggestingIL-15 has anabolic activities and increases skeletal muscle mass (Quinnet al., 1995).

Activated CD4⁺ T lymphocytes play a key role in the development of aneffective immune response against pathogens by providing the growthfactors necessary for the expansion of the activated CD4⁺ T lymphocytes(autocrine growth) and for the expansion of CD8⁺ cytolytic cells and thedifferentiation of B cells into antibody-secreting plasma cells(paracrine “helper” activity).

After clearance of the pathogen, a subfraction of the generatedAg-specific T cells persist as memory cells, either in the lymphoidtissue or in the circulation. Throughout this application, “memorycells” are defined as antigen-experienced cells. These memorylymphocytes are small, resting cells which are optimally primed for thegeneration of a quantitatively and qualitatively superior, secondaryresponse upon a re-encounter with the priming Ag. In order to accomplishthe transition from activated CD4⁺ effector cell to resting CD4⁺ memorycell and to acquire long-term survival, these effectors need to acquirethe following characteristics:

(i) being resistant towards, or escaping from, activation-induced celldeath (AICD); AICD is responsible for attenuation of the immunereaction;

(ii) being independent from autocrine growth factors, produced duringthe immune response. Normally, the disappearance of these growthfactors—a consequence of the ending of immune activity—results in growthfactor depletion-induced cell death by apoptosis;

(iii) having the capacity, in case of a renewed contact with theantigen, to expand maximally by production of the necessary autocrine-and paracrine-acting helper cytokines such as IL-2.

SUMMARY OF THE INVENTION

The research that resulted in the present invention, indicated thatIL-15 promotes the generation and persistence of CD4⁺ memory cells, bypromoting antigen activated CD4⁺ T-lymphocytes to acquire thecharacteristics, mentioned above: resistance towards AICD, insensitivitytowards apoptosis following growth factor withdrawal at the end of theantigen stimulus and high responsiveness towards renewed antigenchallenge. Resistance towards AICD and insensitivity towards apoptosisdetermine the survival of the CD4⁺ T lymphocytes. Responsiveness ischaracterised by cell division, expansion of the cell number andproduction of helper cytokines.

Thus, treatment of antigen stimulated CD⁺ cells with IL-15, even at verylow concentrations, turns off the program of cell death running in theabsence of growth factor. Unlike with IL-2, survival of CD4⁺ T cellswith IL-15 is not accompanied by DNA synthesis nor proliferation,demonstrating that IL-15 induces a resting phenotype in these cells.Moreover, the sensitivity towards AICD of CD4⁺ T lymphocytes, culturedin presence of IL-2, is reversed by IL-15. Restimulation of these IL-15treated, resting T cells with a suitable antigen (Ag) presented by Agpresenting cells (APC) results in maximal cell expansion, driven by arenewed production of helper cytokines. This cell expansion is notattenuated by a massive cell death as a consequence of AICD. In contrastto what is observed for cells cultured in presence of IL-2, theabove-mentioned activities of IL-15 provide a method to achieve survivalof immuno competent CD4⁺ T lymphocytes, herewith strongly improving thesecondary restimulation of CD4⁺ T lymphocytes. In other words, theformation of immunological CD4⁺ memory cells can be controlled in apositive sense, by an increased IL-15 activity, or in a negative sense,by a decreased IL-15 activity.

A first aspect of the present invention thus relates to the use of IL-15in the manufacturing of a pharmaceutical preparation for the stimulationof the formation of memory cells. Such a stimulation can be used in anumber of applications. It can be applied before, during or aftervaccination to increase the efficiency of the vaccination againstinfection or diseases of which the pathological evolution derives, atleast in part, from an inadequate CD4⁺ T cell-dependent immune response.Thus, diseases, where the existence of sufficient numbers of Ag-specificCD4⁺ memory cells is necessary to control (re-emergent) pathogens, aresuitable indications for IL-15 treatment. Important but non-limitingexamples of such pathogenic conditions are bacterial, parasitical orviral infections (e.g. HIV) and cancer.

Other possible indications of this approach are individuals showinghyporesponsiveness towards pathogens or vaccins, or suffering from achronic infection or from a generally weakened immune condition. As weassume that the action of IL-15 becomes even more important towards theend of an acute immune response, promoting the subsequent quiescentperiod, therapeutic doses of IL-15 should preferentially be administeredwhen the immune response is subsiding, in this way favouring theestablishment and long-term survival of CD4⁺ memory cells.

A second aspect of the present invention relates to those cases where anunwanted or harmful CD4⁺ T cell-dependent immune response is(co)-responsible for disease. As an example, several reportsdemonstrated the involvement of autopathogenic CD4⁺ T cells inautoimmune conditions. As a consequence it is anticipated that blockingthe activity of IL-15 will suppress the long-term survival ofautoreactive CD4⁺ effector T cell clones as well as promote theregression of already formed autoreactive CD4⁺ T cells, thus resultingin beneficial effects for patients suffering from an auto-immunecondition. Therapy aiming at the inhibition of IL-15 activities can beaccomplished by administration of agents interfering with the binding ofIL-15 to its receptor such as antagonistic anti-IL-15 antibodies oranti-IL-15Rα antibodies or the Fab or F(ab')₂ fragments of these Ab,soluble IL-15Rα, fusion proteins consisting of soluble IL-15Rα and Fcfragment, or peptides binding with high affinity on the IL-15Rα withoutinducing signalling. A different approach consists of inhibiting IL-15synthesis by administration of IL-15 antisense oligonucleotides throughdirect vaccination of patients with naked DNA, or by gene therapyapproaches.

A third embodiment of the invention further relates to a pharmaceuticalpreparation promoting the formation of memory cells, which preparationcontains IL-15 or IL-15 promoting compounds, possibly in presence of asuitable excipient. A fourth embodiment of the invention relates to apharmaceutical preparation inhibiting the formation of memory cells,which preparation contains IL-15 inhibiting and/or eliminatingcompounds, such as IL-15 antibodies or compounds that interfere with thebinding of IL-15 with its receptor, such as soluble IL15Rα, possibly inpresence of a suitable excipient.

For the use of IL-15 according to the present invention, IL-15 can beadministered by bolus injection, continuous infusion, sustained releasefrom implants or other suitable technique. Administration may be byintravenous injection, subcutaneous injection, or parenteral orintraperitoneal infusion. IL-15 therapeutic agent will be administeredin the form of a pharmaceutical composition comprising purifiedpolypeptide in conjunction with physiologically acceptable carriers,excipients or diluents. Such carriers will be nontoxic to patients atthe dosages and concentrations employed. Ordinarily, the preparation ofsuch compositions entails combining a mammalian IL-15 polypeptide orderivative thereof with buffers, antioxidants such as ascorbic acid, lowmolecular weight (less than about 10 residues) polypeptides, proteins,amino acids, carbohydrates including glucose, sucrose or dextrans,chelating agents such as EDTA, glutathione and other stabilizers andexcipients. Neutral buffered saline or saline mixed with conspecificserum albumin are exemplary appropriate diluents. Elevated levels ofIL-15 can also be obtained by adoptive transfer of cells ex vivotransfected with constructs consisting of an IL-15 cDNA sequence drivenby a potent promoter, or by introduction into the target cells of anIL-15 cDNA sequence after a suitable promoter.

The meaning of therapeutic in the present application is not limited tothe treatment of an existing disease or condition, but comprises the useof IL-15 as support during vaccination and other profylactivetreatments, where the formation of immunological memory cells isessential or helpful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: IL-15 is mainly a survival factor, while IL-2 is mainly aproliferation-inducing factor.

T-HA cells were harvested on day 4 after antigenic restimulation, andwere incubated in 200 μl containing the indicated concentrations of IL-2or IL-15 for 72 h.

(A) l×10⁴ T-HA cells were cultured for 72 h with increasingconcentrations of IL-2 or IL-15. Cultures were pulsed with ³H-thymidinefor the last 8 h.

(B) Viable cell numbers in micro-cultures seeded with 2.10⁴ T-HA cellswere determined on day 3 of culture by trypan-blue dye exclusion.Results shown are averages of 2 haemocytometer counts of 2 wells.

(C) % apoptotic cells in the source cultures was determined by flowcytometric quantitation of cells, which had taken up the exclusion dyePI.

FIG. 2: IL-15 induces a resting phenotype

2×10⁴ T-HA cells were cultured with IL-2 (10 ng/ml) or IL-15 (1 ng/ml)for 48-72 h. Cell cycle status, cell size and expression of activationmarkers were analysed. (A) PI fluorescence intensity as a measure ofcellular DNA content, and cell cycle distribution percentages. (B)forward light scatter as a measure of cell size. (C) (D) CD25 and CD71expression. Dotted lines represent labeling with secondary Ab alone. (E)Rhodaminel23 incorporation indicative for mitochondrial membranepotential values (MFI: mean fluorescence intensity).

FIG. 3: IL-15 or low-dose IL-2 treated T-HA cells show increasedproliferation in response to Ag/APC stimulation.

T-HA lymphocytes were harvested on day 12 after antigenic restimulationand cultured for 48 h with the indicated concentrations of IL-2 or IL-15in 24-well plates. Next, pretreated cells were harvested, cytokine waswashed away and 1×10⁴ cells were stimulated with 2×10⁵ irradiated spleencells as APC and 200 ng/ml purified BHA as antigen. ³H-thymidine wasadded for the last 12 h of the 84 h culture period. Data are expressedas cpm of ³H-thymidine incorporated.

FIG. 4: IL-15 or low-dose IL-2 treatment of T-HA cells results inincreased generation of effector cells during an Ag/APC response.

T-HA lymphocytes were harvested on day 12 after antigenic restimulationand were pretreated for 48 h with IL-2 (7.7 ng/ml or 0.077 ng/ml) orIL-15 (1 ng/ml) followed by labelling with the green fluorescentmembrane marker PKH2-GL. 12 h later, 1×10⁴ labeled cells were stimulatedwith 2×10⁵ irradiated spleen cells as APC and 200 ng/ml purified BHA asantigen. Cultures were harvested at the indicated time points and dead(A) and viable (B) cell numbers of the stained cell population weredetermined by flow cytometry and PI uptake, using the unlabeled spleencells as an internal standard. Countings shown are averages oftriplicate cultures.

FIG. 5: Culture of T-HA cells in the presence of IL-15 results both inoptimal recovery of cells after primary antigenic restimulation and inoptimal proliferative responsiveness upon secondary antigenicrestimulation.

1×10⁵ T-HA lymphocytes, pretreated for 48 h with IL-2 (10 or 0.1 ng/ml)or IL-15 (1 ng/ml), were stimulated in 1 ml with 2×10⁶ irradiated spleencells and 200 ng/ml BHA. On day 4 these cultures were supplemented withthe same concentrations of cytokine as during the pretreatment andincubated under these conditions for 8 more days.

(A) Viable cells were counted by trypan blue dye exclusion on day 12after starting the antigenic restimulation described above. Data arerepresented as a cell recovery index, i.e. the factor by which the inputcell number has multiplied on day 12.

(B) 1×10⁴ of the recovered cells were stimulated a second time withAg/APC and proliferation was determined by ³H-thymidine incorporation.

(C) The total proliferative response expected during the secondantigenic restimulation is represented as a reactivity index, calculatedas follows:(cell recovery index described in FIG. 5(A)).times.(CPM measured on day15 (FIG. 5(B))

FIG. 6: Culture of T-HA cells in the presence of IL-15 results in astrongly enhanced generation of immune effector cells.

Starting from day −2, T-HA lymphocytes were pretreated for 48 h with theindicated concentrations of IL-2 or IL-15. On day 0, 1×10⁵ T-HA cellswere stimulated in 1 ml with 2×10⁶ irradiated spleen cells and 200 ng/mlBHA. On day 4 these cultures were supplemented with the sameconcentrations of cytokine as during the pretreatment and incubatedunder these conditions for 8 more days. On day 12 after the firstantigenic stimulation, 1×10⁴ cells were restimulated with Ag/APC and thenumber of generated effectors was determined on day 15 as previouslydescribed. The total number of effector cells generated on day 15 percell stimulated on day 0 is represented as a cell recovery indexcalculated as follows: $\frac{\begin{matrix}{\left( {{number}\quad{of}\quad{cells}\quad{harvested}\quad{from}\quad{the}\quad{cultures}\quad{on}\quad{day}\quad 12} \right) \times} \\\left( {{number}\quad{of}\quad{cells}\quad{counted}\quad{on}\quad{day}\quad 15} \right)\end{matrix}}{10^{5} \times 10^{4}}$

FIG. 7: IL-15 protects activated polyclonal CD4⁺ T cell populationsagainst growth factor withdrawal-induced PCD and TCR-induced death.

Freshly isolated, unsorted spleen cells from C57BI/6 mice werepolyclonally activated with soluble anti-CD3 mAb (1 μg/ml). On day 4,CD4⁺ T cells were isolated by magnetic cell sorting and 7.5×10⁶ cellswere further cultured for 10 days without exogenously added cytokine orwith addition of IL-15 (10 ng/ml) or IL-2 (10 ng/ml). On day 14 afterthe initial stimulation, cultures were harvested and survival,sensitivity for TCR-induced death and TCR-responsiveness were evaluated.(A) Viable CD4⁺ T cell numbers were counted after addition of trypanblue. Survival is presented as the percentage recovery of the input cellnumbers. 3 independent countings were performed, SD<15%. (B)Susceptibility for TCR-induced death was evaluated by restimulation of1×10⁴ viable IL-15- or IL-2-cultured cells, isolated by density gradientcentrifugation, with plate-bound anti-CD3 mAb (10 μg/ml) for 24 h anddetermination of percentages of apoptotic CD4⁺ T cells by PI uptake.Results represent 3 pooled wells. (C) Secondary responsiveness ofactivated CD4⁺ T cell populations to appropriate TCR-stimulation wasmeasured by restimulating 1×10⁴ pretreated T lymphocytes with 1 μg/mlsoluble anti-CD3 mAb and 2×10⁴ IFN-.gamma.-activated macrophages(Mf4/4), either in the absence or presence of 1 ng/ml IL-15 or IL-2.Naive CD4⁺ T cells were added as a control to assure that thesestimulation conditions could properly induce a proliferative response.Proliferation was measured by addition of ³H-thymidine for the last 12 hof the 84 h assay period. No proliferation could be detected in culturesof T cells and Mf4/4 without soluble anti-CD3 Ab (cpm<500) indicatingthat the observed response was strictly dependent on TCR triggering.Results represent means of triplicate cultures. Experiments on freshlyisolated spleen cells were done 2 times with similar results.

FIG. 8: In vivo administration of IL-15 during/after a primary antigenicchallenge augments proliferative reponsiveness of primed LN cells to asecondary antigenic challenge.

Mice were immunized with HA and treated for 14 days with IL-15 or PBSdelivered by an ALZET mini-osmotic pump. On day 21 after the initial HAinjection, mice were sacrificed and draining lymph nodes prepared. 2×10⁵LN cells were restimulated in 96-well microtiter plates with 500 ng/mlHA or HEL without exogenous cytokine. Results show the HA-specificproliferation, i.e. absolute HA-induced proliferation minus HEL-inducedproliferation, of LN cell cultures from IL-15- (hatched) or PBS-treated(dotted) mice, measured by addition of ³H-thymidine after 72 h (A) or120 h (B) for an additional 12 h. Data shown in (C) also representHA-specific proliferation at 120 h but here 1 ng/ml exogenous IL-15 wasadded during the culture period. Each bar represents proliferation of LNcells derived from one individual mouse, except for the naive mice(black) where LN of three individuals were pooled.

FIG. 9: In vivo administration of IL-15 during/after a primary antigenicchallenge augments Ag-specific Ab titers elicited by a secondaryantigenic challenge. Mice were immunized with HA and treated for 10 dayswith IL-15 or PBS delivered by daily bolus injections. One day after thelast delivery, IL-15-or PBS-treated mice were rechallenged with HA. Twoweeks later, blood samples were taken, sera prepared and anti-HA Abdetected with an indirect ELISA. The sera were serially diluted inMaxisorp 96-well plates (Nunc, Roskilde, Denmark) previously coated withHA by overnight incubation at 4° C. with 0,5 μg/ml stock solution of theAg. Bound Ab was detected with goat anti-mouse IgG Ab (Sigma) usingalkaline phosphatase-conjugated rabbit anti-goat IgG as detecting Ab(Sigma). Results are represented as O.D. values as a function of serumdilution factor. Each curve represents serum of an individual animal.Sera of naive mice were used as a control on background signals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention can be executed with isolated IL-15. SuitableIL-15 sources are the culture medium of constitutively IL-15 producinghuman cell lines such as the T-102. Alternatively, recombinant IL-15 canbe applied. WO 95/27722 gives the information needed to preparerecombinant IL-15. Recombinant IL-15 is commercial available as well.Alternatively, variants of IL-15 can be used, as long as they have theactivity needed to stimulate the formation of memory cells. Thesevariants are identified as “active variants”. Active variants furthercomprise IL-15 fragments displaying sufficient IL-15 activity to beuseful in the invention. Moreover, the activity of IL-15 can bestimulated in an indirect way by the addition of IL-15 inducingcompounds, such as LPS for induction of IL-15 production in monocytes,or by IL-15 inducing methods, such as UVβ irradiation for keratinocytes.

For the second variant of the invention, isolated IL-15 inhibiting oreliminating compounds can be used. If these compounds are(poly)peptides, they may be produced by recombinant DNA techniques.

The present invention will be further elucidated with reference to theexample below, which is only intended by way of explanation and does notimply any limitation whatever to the scope of the invention.

EXAMPLE

1. Materials and Methods

1.1. CD4⁺ T Cell Clone

The influenza A/H3 haemagglutinin (HA)-specific and H-2^(b) restrictedCD4⁺ murine T cell clone T-HA was developed in this laboratory by aninitial immunisation of C57BI/6 mice with 100 μg/ml bromelain cleavedhaemagglutinin (BHA) and 0.5 mg/ml adjuvant (Ribi, Immunochem ResearchInc., Hamilton, Mont., USA) and a second immunisation with 32 μg/ml BHAthree weeks later.

5 days after this boost immunisation lymph nodes were isolated and 3.10⁷cells were stimulated in vitro with 0.5 μg/ml BHA in 25 cm² cultureflasks (Nunclon, Nunc, Roskilde, Denmark). On day 4 10 U/ml mouse IL-2(from PMA stimulated EL4.IL-2 cells) was added to the cultures.

After 2 additional biweekly restimulations with 0.5 μg/ml BHA and APC, apool of optimally HA-reactive T-lymphocytes was obtained. These T-HAcells were maintained long term in vitro by biweekly restimulation in 25cm² culture flasks with 10 ng/ml BHA and 7×10⁷ syngeneic spleen cells(3000 rad gamma irradiated). On day 2, 30 IU/ml rhIL-2 was added and Tcells were further cultured and expanded by medium renewal and IL-2addition every 4 days. C57BI/6 mice (Broekman Instituut, Eindhoven,Netherlands) were used as a source of spleen cells. T-HA cells werecultured in 12.5 mM Hepes-buffered RPMI 1640 medium (Life Technologies,Paisley, Scotland) supplemented with 10% FCS (Life ScienceInternational), 2 mM Glutamax-I, penicillin/streptomycin, 1 mM sodiumpyruvate (all from Life Technologies, Paisley, Scotland) and 5×10⁻⁵ M2-ME (BDH, Poole, England).

1.2. Cytokines

Recombinant human IL-2 (r-IL-2) had a specific activity of 1.3×10⁷ lU/mgas determined in the CTLL-2 assay, hence 1 IU corresponds to 77 pg.

Recombinant human IL-15 (r-IL-15) was purchased from PeproTech (London,UK) and had a specific activity of 2×10⁶ U/mg according to themanufacturer.

Hereafter, ‘rIL-2’ and ‘IL-2’, as well as ‘r-IL-15’ and ‘IL-15’ are usedinterchangeably because it is not essential for the invention to use arecombinant form.

1.3. IL-2 or IL-15 Pretreatment

T-HA cells were harvested from cultures by incubation in non-enzymaticcell dissociation buffer (Sigma) and viable cells were separated fromremaining irradiated spleen cells and dead cells by centrifugation on aHistopaque-1077 (Sigma, Irvine, UK) density gradient for 25 min at 2000rpm. 2-5×10⁵ T-HA cells were cultured for 48 h in 24-well flat-bottomtissue culture plates (Falcon) in the presence of variableconcentrations of IL-2 or IL-15.

1.4. Proliferation Assays

Induction of proliferation by IL-2, IL-15 or Ag/APC was measured byincubating 1×10⁴ T-HA cells with serial dilutions of IL-2, IL-15 or 200ng/ml BHA and 2×10⁵ irradiated C57BI/6 spleen cells as a source of APCin 96-well, flat-bottom, microtiter plates (Falcon 3072, BectonDickinson, Franklin Lakes, N.J., USA). ³H-thymidine (Amersham) was addedat 0.5 μCi/well for the last 8-12 h of the indicated incubation period.Cells were harvested on glass fiber filters and ³H-thymidineincorporation was measured on a Topcount betaplate counter (Packard).Results reported are means of triplicate cultures.

1.5. Cell-labelling with PKH2-GL

T-HA cells were harvested and washed twice in medium devoid of serum inpolypropylene tubes. 1×10⁶-1×10⁷ cells were resuspended in 1 ml diluentA and stained with 2 μM PKH2-GL (Sigma, St. Louis, Mo., USA) followingthe instructions of the manufacturer. Stained cells were washed twicewith medium supplemented with 18% fetal calf serum (FCS) and incubatedovernight in their culture medium to allow dissociation of excess dyefrom the membrane.

1.6. Analysis of Viable and Dead Cell Populations

Viable cell numbers were determined by counting trypan-blue dyeexcluding cells in a hemocytometer. Duplicate wells were always countedtwice and results shown are averages of these 4 independent countings.Apoptosis was analysed by addition of 30 μM propidium iodide (PI) (ICN)to harvested cells and percentage of cells taking up PI was measuredwith an EPICS 753 flow-cytometer (Coulter Electronics, Luton, UK),equipped with an Argon ion laser emitting at 488 nm, after gating outcell debris. PI fluorescence was detected at 610-630 nm. Additionally,percentage of apoptotic cells was also determined by forward scatteranalysis (results not shown). Data obtained by the latter methodcorrelated well with the PI dye exclusion data.

In mixed cultures of PKH2-GL stained T-HA cells and APC splenocytes,numbers of viable and apoptotic T-HA cells were obtained byflow-cytometric analysis of PI-negative and -positive cells respectivelythat emitted green fluorescence (525 nm) from the PKH2-GL stain.

1.7. Antibodies and Reagents

For immunofluorescence, rat anti-mouse CD25 (clone PC 61) and ratanti-mouse CD71 (clone R217 17.1.3, kindly provided by Dr. G. Leclercq)were used as primary antibodies. Anti-CD25 and anti-CD71 binding wasdetected with a FITC-conjugated goat anti-rat IgG (Sera-Lab, CrawleyDown, UK). The mitochondrial membrane potential was measured by additionof 1 μM Rhodaminel123 (Molecular Probes Inc., Eugene, Oreg.) for 30 minto the cells and subsequent flow-cytometric analysis of the fluorescenceintensity.

1.8. Cell Cycle Analysis

T-HA cells were harvested, washed once in cold PBS, and lysed inKrishan's reagent (0.05 mg/ml PI, 0.02 mg/ml ribonuclease A, 0.3%Nonidet P-40, 0.1% sodium citrate). Cell nuclei were analysed for DNAcontent by flow-cytometry. The distribution of cells along the distinctstages of the cell cycle was calculated with MDADS Paral software(Coulter Electronics).

1.9. Experiments with Freshly Isolated Spleen Cells

8×10⁸ spleen cells were prepared from spleens of naive, 8 week oldC57BI/6 mice and were activated in 25 cm² tissue culture flasks (Falcon,Becton Dickinson) with 1 μg/ml soluble anti-CD3 mAb (145-2C11). After 24h, excess antibody was removed and cells were further cultured for 72 hwithout addition of exogenous cytokine. Following this stimulationperiod, cultures were harvested and CD4⁺ T cells were isolated byimmunomagnetic cell sorting. A negative selection procedure, using an Abcocktail designed for the enrichment of murine CD4⁺ T cells (StemSep,Stem Cell Technologies, Vancouver, Canada), was followed according tothe manufacturer's instructions. 7.5×10⁶ recovered cells were furthercultured for 10 days and supplemented (every fourth day) with theirrespective cytokines (no, 10 ng/ml IL-15 or 10 ng/ml IL-2). Viable cellnumbers were determined on day 14 based on trypan blue dye exclusion.For restimulation 1 μg/ml soluble anti-CD3 mAb and the immortalizedmacrophage cell line Mf4/4 (freely available from De Smedt, UniversiteitGent) were used. Prior to use, Mf4/4 cells were activated for 24 h with400 U/ml IFN-γ to enhance expression of costimulatory molecules. Then,they were treated for 90 min with 30 μg/ml Mitomycin-C (Duchefa,Haarlem, The Netherlands) in order to block their proliferation, thusavoiding interference with proliferation-measurements from therestimulated lymphocytes. Alternatively, for determination ofsusceptibility to anti-CD3-induced death, freshly isolated, unsortedspleen cells were activated for 72 h in 24-well plates with 1 μg/mlsoluble anti-CD3 mAb (145-2C11) without exogenous cytokine and weresupplemented on day 3 with 10 ng/ml IL-15 or IL-2. After an additional 8day culture period, the cells were harvested and restimulated withplate-bound anti-CD3 mAb (10 μg/ml). Apoptotic cell numbers weredetermined after 24 h by PI dye uptake. CD4/CD8 ratios were determinedby labeling 1×10⁵ cells with 0.5 μg PE-conjugated rat anti-mouse CD4 mAb(Pharmingen, San Diego, Calif.) and 0.5 μg/ml FITC-labeled ratanti-mouse CD8 mAb (clone 53-6.7, kindly provided by Dr. G. Leclercq,Ghent, Belgium) and, after gating out dead cells and debris, analysis ofstained populations on a FACScalibur flow cytometer (Becton-Dickinson).Absolute numbers of. CD4⁺ T cells in the respective cultures werecalculated from the percentages obtained and total viable cell countingsby trypan-blue dye exclusion.

2. Results

2.1. IL-15 is a Survival Factor, Protecting CD4@+ T Cells Against CellDeath Following Growth Factor Withdrawal without Inducing Proliferation

During the acute activation phase by antigen, CD4⁺ effector cellstransiently produce high levels of IL-2, resulting in autocrine growthand strong expansion of the lymphocytes. However, following terminationof the effector phase and hence, the ending of cytokine production, thegenerated lymphocytes become dependent on the exogenous supply of growthfactors, mainly IL-2, for their survival. We studied the survival ofactivated T-HA cells, harvested four days after stimulation with Ag/APC,in the presence of increasing concentrations of IL-2 or IL-15. Afterthree days of treatment with different concentrations of cytokines, theuptake of ³H-thymidine as a measure for cell division, the absolutenumbers of viable lymphocytes and the percentage apoptotic cells in thevarious cultures were determined (FIG. 1).

In contrast to treatment with IL-2, treatment with IL-15 did not resultin cell division (FIG. 1A). These results are in contrast with theresults obtained by Kanegane & Tosato (1996), who claimed that CD4⁺cells proliferate upon addition of IL-15. This discrepancy is probablydue to the presence of low amounts of IL-2 (besides IL-15) in theirexperiments. Unexpectedly, notwithstanding the absence of growth factoractivity by IL-15, IL-15 treated T-HA lymphocytes showed protectiontowards cell death, normally occurring in absence of growth factoractivity:the cell numbers remained unchanged at an IL-15 concentrationof 0.1 ng/ml or higher (FIG. 1B) and no increase of the percentage ofapoptotic cells was seen (FIG. 1C). This is in clear contrast with theexplicit decrease of the number of viable cells and the correspondingincrease of dead cells in absence of cytokine, a reflection of thegrowth factor depletion-induced cell death as well as with the cellgrowth-associated survival of IL-2 treated T-HA lymphocytes (FIG. 1).

From these data we conclude that IL-15, but not IL-2, is capable ofinducing a survival signal in CD4⁺ T lymphocytes without driving thecells into the proliferative cell cycle. In this way, IL-15 establishesa resting phenotype in T-HA lymphocytes that is linked, however, tosurvival. Moreover, under conditions of low cytokine concentration, inthe range of 0.1 ng/ml, the net recovery of viable T-HA cells wasconsiderably higher when cells were cultured with IL-15 instead of IL-2.Based on the preservation of viability in absence of active celldivision upon IL-15 treatment, this example shows that the IL-15polypeptide gives to the antigen is activated CD4⁺ T lymphocytes theproperty to survive as a resting cell in absence of cytokine withinherent growth activity. In this way, IL-15 acts as a factor, enablingthe survival of antigen-primed CD4⁺ T lymphocytes, generated by apreceding antigenic activation, or resting cells.

2.2. IL-15 Induces a Resting Phenotype

It is thought that after conclusion of a primary immune response afraction of activated effector cells reverts to a resting state andpersists in the animal as a population of small lymphocytes, ready for a“memory” response in case of re-emergency of their antigen. It waswondered whether T-HA lymphocytes surviving with IL-15 without cyclingcould be phenotyped as small, resting lymphocytes. Therefore a number offeatures generally recognized as parameters for lymphocyte quiescencewere studied. It was determined whether the observed growth-arrest tookplace in a specific phase of the cell cycle. Cell cycle analysis by flowcytometry revealed that IL-15 treated cells accumulated in G₀/G₁ (FIG.2), indicative of the induction by IL-15 of an arrest in cell cycleentry. Thus, cycling cells treated with IL-15 are neither arrestedimmediately nor randomly, which in fact would be apoptosis-inducing, butproceed with their cycle until they reach G₀/G₁ and then exit cell cycleprogression in an orderly manner, i.e. without triggering programmedcell death.

Additionally, cell size, expression of activation markers andmitochondrial transmembrane potential (ΔΨm) as an indicator of themetabolic state of the cells were evaluated. IL-15-treated T-HA cellsexhibited all the hallmarks of resting cells: the cells were small,expressed low levels of the CD25 (IL-2Rα) and CD71 (transferrinreceptor) activation markers, and had a low ΔΨm (FIG. 2). In contrast,IL-2-cultured cells were large blastoid cells with high CD25 and CD71expression levels and a high oxidative metabolism as indicated by theincreased ΔΨm. Thus, the IL-15-induced arrest in G₀/G₁ of T-HA cells isaccompanied by acquisition of a typical quiescent phenotype.

2.3. IL-15 Protects the CD4⁺ Helper T Cell Clone T-HA Against AICD

AICD of mature T-lymphocytes is generally considered as a key mechanism,restricting both the strength and duration of an immune response(Critchfield et al., 1995). It has been shown that clonal expansion andas a consequence, IL-2 is an important regulator of susceptibility toAICD as T lymphocytes cultured in the presence of IL-2 easily undergoapoptosis following crosslinking of the T-lymphocyte receptor forantigen (TCR) (Lenardo, 1991). We compared the influence of IL-2 andIL-15 treatment on responsiveness of T-HA cells towards stimulation byAg/APC. T-HA cells were treated for 48 h with IL-15 (1 ng/ml) or IL-2(100 lU/ml (7.7 ng/ml) or 1 IU/ml (0.077 ng/ml)) prior to antigenicrestimulation.

As was described above, cells differed functionally at the moment ofrestimulation according to the cytokine added: IL-15 kept the T-HA cellsfully viable but non-proliferating, while high-dose IL-2 (7.7 ng/ml)induced vigorous cell cycling and treatment with low-dose IL-2 (0.077ng/ml) resulted in extensive cell death. T-HA cells pretreated withIL-15 proliferated dramatically stronger in response to antigenicrestimulation as compared to cells cultured in the presence of 100 IUIL-2 (FIG. 3). The small fraction of T-HA cells surviving incubationwith 1 IU/ml IL-2, exhibited a reactivity towards Ag/APC similar tocells pretreated with IL-1 5.

In a next step we wanted to answer the question whether thisdifferential responsiveness was a reflection of a differentialsensitivity towards AICD or rather the consequence of differingproliferative capacities of IL-2 versus IL-15 pretreated cells. For thispurpose, T-HA cells were labelled with a green fluorescent dye prior toAg/APC stimulation, allowing them to be discriminated from APC presentin these cultures during analysis of viable and dead cell numbers byflow cytometry.

The results shown in FIG. 4A clearly demonstrate that the defectiveimmune responsiveness observed with T-HA cells pretreated with 7.7 ng/mlIL-2 is the consequence of extensive cell death, apparent 48 h afterstart of the activation with Ag/APC. This is in agreement with thegenerally accepted view that IL-2 sensitizes T cells for AICD. On thecontrary, T-HA cells cultered in the presence of IL-15 were efficientlyprotected against cell death occurring during antigenic activation,resulting in the generation of high numbers of effector cells (FIG. 4).Comparable results were obtained with the small population of T-HA cellssurviving a low-dose 0.077 ng/ml IL-2 treatment. This result illustratesthe inhibitory activity of IL-15 on Ag/APC induced cell death (AICD)and, as a result of this activity, the considerably increased formationof effector T-lymphocytes after a renewed, secondary stimulation withantigen of the IL-15 treated, resting CD4⁺ T lymphocytes.

2.4. Protection by IL-15 Against AICD and Against Growth FactorWithdrawal-induced Apoptosis Strongly Potentiates Immune ResponsivenessUpon Rechallenge with Antigen

In this example, the effect of the IL-15 treatment on the strength ofthe secondary immune response of CD4⁺ T lymphocytes is illustrated. T-HAlymphocytes were cultured in the presence of IL-15, or in high- orlow-dose IL-2 before and after restimulation with Ag/APC.

As shown in FIG. 5A, the presence of IL-15 resulted in the preservationover a long time of the highest number of T-lymphocytes, generated inresponse to a preceeding antigenic stimulation. Thus, starting from afixed number of IL-15 treated T-HA cells, the combination of optimalproliferation in response to Ag/APC stimulation and optimal persistenceof the generated effector cells by addition of IL-15 after terminationof the response to antigen, resulted in a 31-fold increase of T cellsavailable for a second Ag/APC response. High-dose IL-2, sensitizingtowards AICD, or low-dose IL-2, insufficiently supporting growth andviability, raised T cell numbers 27.3 and 6.7 fold respectively (FIG.5A).

T cells that survive with IL-15 remained optimally sensitive towardssubsequent activation by antigen (FIG. 5B). The combination of a highyield of T lymphocytes after primary stimulation with antigen, optimalpersistence and high reactivity upon renewed stimulation with antigen ofIL-15 treated CD4⁺ T lymphocytes resulted in a maximal reactivity indexof the thus treated T-HA cells (FIG. 5C). Furthermore, the cumulativeeffect of this reactivity leads to a 105 fold yield in effector cells asshown in FIG. 6. T-HA lymphocytes that survive due to high or low dosesof IL-2 generated a considerably weaker reactivity index (FIG. 5C) and,as a consequence, merely 20 and 8 fold increases in cell numbers,respectively, 3 days after secondary stimulation (FIG. 6).

Obviously, IL-15 both enhances the availability as well as theresponsiveness of cells resulting in maximal secondary responses,features that cannot be achieved by any dose of IL-2. These resultsclearly demonstrate that IL-15, but not IL-2, possesses the propertiesnecessary for generating an efficient immune memory, i.e. providing astrong survival signal allowing the persistence of immune effectors in aquiescent state and priming these memory cells for optimal response incase of renewed Ag stimulation by inducing insensitivity towards AICD.Taken together our study defines a unique novel pro-memory function forIL-15, clearly distinct from the physiological activities exerted byIL-2 or other cytokines with growth factor activity.

2.5. Induction of Quiescence and Protection Against Apoptosis by IL-15Also Occurs with ex vivo Isolated T Cells

Fresh, unsorted spleen cells from naive C57BI/6 mice were isolated andpolyclonally stimulated in vitro. The stimulus consisted of solubleanti-CD3 mAb (1 μg/ml) which, in the presence of costimulation by spleenAPC, polyclonally activates naive T cells (Tamura, T., and Nariuchi, H.,J. Immunol. 148:2370, 1992). After 24 h, remaining anti-CD3 mAb wasremoved and the activated cells were further cultures in the absence ofexogenous cytokine. To confirm that activation occurred, anti-CD3mAb-activated and unstimulated cells were pulsed with ³H-thymidine.Soluble anti-CD3 mAb induced a strong proliferative response: 25,304 cpmas opposed to 2,581 cpm for unstimulated cells. On day 4, CD4⁺ cellswere isolated by immunomagnetic cell sorting and further culturedwithout cytokine or in presence of IL-15 (1 ng/ml).

After 10 days of culture in the absence of exogenous cytokine, viablecell numbers had dropped to 15% of the cell input, while IL-15maintained cell numbers at approximately 60% of cell input (FIG. 7A).Cells surviving with IL-15 appeared as small resting lymphocytes and didnot reveal DNA synthesis (59 cpm with 10 ng/ml IL-15) whereasproliferation could be induced with IL-2 (4,184 cpm with 10 ng/ml).Hence, also for freshly isolated and TCR-activated CD4⁺ T cells, IL-15acts as a survival factor and induces quiescence.

Next, the resistance to TCR-induced cell death was investigated,triggered by immobilized anti-CD3 mAb, in these polyclonally activated Tcell cultures. The CD4⁺ T cell population maintained throughout withIL-15 was largely resistant, whereas cells cultured with IL-2 showedextensive cell death (FIG. 7). Finally, CD4⁺ T cells residing in anIL-15-induced, resting state proliferated in response to renewedstimulation with soluble anti-CD3 and APC, while cells maintained withIL-2 did not (FIG. 7C). Also here, addition of IL-15 to the IL-15pretreated cultures further increased the proliferative response, henceconfirming the growth-promoting activity of IL-15 in the presence of TCRaggregation. These experiments demonstrate that the characteristicsinduced by IL-15 in the clonal CD4⁺ T cell T-HA namely long-termsurvival as a resting population, resistance to apoptosis and increasedresponsiveness to TCR restimulation, are also acquired by freshlyisolated CD4⁺ T cells treated with IL-15.

2.6. In vivo Evaluation of the Capacity of IL-15 to Enhance MemoryResponses

In vitro, the activities of IL-15 described here show that this cytokineis a factor promoting the generation and persistence of memory CD4⁺ Tcells, thus enhancing secondary/memory immune responses to an antigen.It was investigated whether administration of IL-15 in vivo duringand/or after a primary immune response against an antigen could enhancethe secondary/memory response against this antigen.

Therefore IL-15 was delivered by a slow-release mini-osmotic pump or bybolus injections to mice immunized with haemaglutinnin (HA) andresponsiveness to a renewed challenge with this antigen was evaluated.In the first experiment the initial immunisation consisted of twoinjections with HA: 5 μg injected intraperitoneal (IP) on day 0 andanother 5 μg injected subcutaneous (SC) in the right flank on day 3. Onday 4, a bolus injection of 1 μg hIL-15 in a volume of 100 μl PBS wasgiven (or 100 μl PBS as a control). Two hours after this initialdelivery, an ALZET mini-osmotic pump (model 2002, Alza Corp., Palo Alto,Calif.), filled with 10 μg hIL-15 in 235 (.+-.5,7) μl PBS or with PBSwithout cytokine, was implanted SC on the back of the mouse. The openingof the pump was oriented towards the place where the SC injection of HAwas given. Filling of the pump was performed according to themanufacturer's instructions. The pumps released IL-15 in the animal at aflow rate of 0,52 (.+-.0,03)μl/hr during 14 days.

On day 21 after the first HA injection, mice were sacrificed and thedraining lymph nodes (LN) of IL-15 or PBS-treated mice were isolated aswell as LN from naive mice, LN cells were prepared and restimulated invitro with 500 ng/ml HA or henn egg lysozyme (HEL) as an irrelevantantigen. Proliferation induced by these antigens was measured by³H-thymidine incorporation. Results shown in FIG. 8 represent thespecific proliferation against HA, i.e. cpm value obtained with HEL issubtracted from the absolute cpm value obtained with HA, from LN cellsof two mice. Proliferation after 72 h (FIG. 8A) as well as 120 h (FIG.8B) of LN cells from IL-15-treated mice was markedly increased, ascompared to PBS-treated or naive mice. Also, addition of exogenous IL-15during the restimulation period could prolong Ag-specific proliferativeresponses (FIG. 8C). Again, LN cells from IL-15-treated mice were mostresponsive to this exogenous IL-15.

The data demonstrate that slow-release delivery of IL-15 during and/orafter a primary immune response results in an augmented proliferativeresponsiveness of cells from the draining LN against the immunizingantigen, indicative for the presence of a higher frequency of memory Tcells in the immunized animal.

In a second experiment, mice were immunized with a single SC bolusinjection of 2,5 μg HA (day 0) at the right flank and were treated,starting on day 4 after this initial immunization, with daily bolusinjections of IL-15 for 10 consecutive days. The following doses wereadministered: day 4, 5 μg IL-15; day 5-7, 4 μg; day 8-10, 3 μg and day11-13, 2 μg. The cytokine was injected SC at the same site as the Ag ina volume of 100 μl PBS. Control mice were injected with PBS withoutcytokine. On day 14, a new challenge of 2,5 μg HA was given to theIL-15- and PBS-treated mice, injected SC at the same site as the initialimmunisation. Two weeks later, blood was collected by retro-orbitalbleeding and serum was prepared immediately by incubating the bloodsamples at 37 DEG C. for 20 minutes. Titers of antibody (Ab) directedagainst HA were determined by indirect ELISA.

FIG. 9 demonstrates that the anti-HA Ab titer of the two IL-1 5-treatedmice was enhanced, as compared to the four PBS treated mice. Titers innaive mice are shown as an additional control on ELISA backgroundlevels. The augmented anti-HA Ab titers are indicative that an enhancedimmune response against the second HA challenge occurred in mice treatedwith IL-15 after the primary response. Taken together, it is shown thatadministration of IL-15 in vivo during and/or after an ongoing immuneresponse enhances the secondary/memory response elicited by a renewedcontact with the Ag involved.

List of Abbreviations

-   aa: amino acid-   Ab: antibody-   Ag: antigen-   AICD: activation-induced cell death-   APC: antigen-presenting cell-   BHA: bromelain cleaved haemagglutinin-   IL: interleukin-   LAK: lymphokine activated killer cell-   2-ME: P-mercaptoethanol-   PBT: peripheral blood T lymphocytes-   PHA: phytohaemagglutinin-   TCR: T cell receptor-   Th: T helper lymphocyte-   cpm: count per minute

REFERENCES

-   1. Armitage R J et al. (1995) J Immunol 154(2): 483-90-   2. Bamford R N et al. (1994) Proc Natl Acad Sci USA 91(11): 4940-4-   3. Burton J D et al. (1994) Proc Natl Acad Sci USA 91(11): 4935-9-   4. Carson W E et al. (1994) J Exp Med 180(4): 1395-403-   5. Critchfield J M et al. in ‘Apoptosis and the immune response’,    1995, Wiley-Liss, Inc., New York; p. 55-114-   6. Giri J G et al. (1995) EMBO J 14(15): 3654-63-   7. Grabstein K H et al (1994) Science 264(5161): 965-8-   8. Gray D (1993) Annu Rev Immunol 11: 49-77-   9. Kanegane H & Tosato G (1996) Blood 88(1): 230-5-   10. Khan I A & Kasper L H (1996) J Immunol 157: 2103-2108-   11. Leclercq G et al. (1996) J Exp Med 184(2): 325-36-   12. Lenardo M J (1991) Nature 353(6347): 858 61-   13. Mueller D L et al. (1996) J Immunol 156(5): 1764-71-   14. Nieto M et al. (1996) Eur J Immunol 26(6): 1302-7-   15. Nishimura H et al. (1996) J Immunol 156(2): 663-9-   16. Puzanov I J et al. (1996) J Immunol. 157: 4282-4285-   17. Quinn L S et al. (1995) Endocrinology 136(8): 3669-72-   18. Swain S L et al (1996) Immunological Reviews 150: 143-167-   19. Tagaya Y et al. (1996) Immunity 4 (4): 329-36-   20. Tamura T and Nariuchi, H (1992) J. Immunol. 148(8): 2370-2377-   21. Wilkinson P C & Liew F Y (1995) J Exp Med. 181(3): 1255-9-   22. Zhang X et al. (1995) J Exp Med 182(3): 699-709

1. A method of suppressing formation of memory cells of the immunesystem of a mammal in need thereof, comprising administering IL-15inhibiting or eliminating compounds to the mammal.
 2. A method accordingto claim 1 wherein the memory cells are CD4⁺ T lymphocytes.
 3. A methodaccording to claim 1 wherein said compound is selected from the groupconsisting of an anti-IL-15 antibody, anti-IL-15Rα antibody, fragmentsof these antibodies, soluble IL-15Rα, fusion proteins comprising solubleIL-1 5Rα and Fc fragment, compounds binding and/or inhibiting functionalIL-15 receptor, and IL-15 antisense oligonucleotides.
 4. A methodaccording to claim 1 for use in the treatment of auto-immune diseases orin treatment before, during or after transplantation.
 5. A methodaccording to claim 1 for use in the treatment of immune deficiencydiseases or in treatment before, during or after vaccination.
 6. Apharmaceutical composition comprising IL-15 or active variants thereofand/or IL-15 activity enhancing compounds, optionally together with asuitable excipient.
 7. The pharmaceutical composition according to claim6, wherein the IL-15 activity enhancing compound is lipopolysaccharide(LPS).
 8. A pharmaceutical composition comprising IL-15 inhibiting oreliminating compounds, optionally together with a suitable excipient. 9.The pharmaceutical composition according to claim 8, wherein thecompound is selected from the group consisting of an anti-IL-15antibody, anti-IL-15Rα antibody, fragments of these antibodies, solubleIL-15Rα, fusion proteins comprising soluble IL-15Rα and Fc fragment,compounds binding and/or inhibiting functional IL-15 receptor, and IL-15antisense oligonucleotides.